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Title of Journal: Plant Mol Biol Rep

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Abbravation: Plant Molecular Biology Reporter

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Springer-Verlag

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DOI

10.1002/jor.1100170622

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1572-9818

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Molecular Cloning Biochemical Characterization a

Authors: Rishi K Vishwakarma Ruby Somesh Singh Prashant D Sonawane Sameer Srivastava Uma Kumari R J Santosh Kumar Bashir M Khan
Publish Date: 2012/11/21
Volume: 31, Issue: 3, Pages: 547-557
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Abstract

Bacopa monniera Brahmi is an important Indian medicinal herb found in wet damp and marshy places It produces medicinally important compounds known as bacosides along with alkaloids like brahmine and herpestine Bacosides are triterpenoid saponins and their biosynthesis takes place via the isoprenoid pathway starting with acetylCoA AcetylCoA Cacetyltransferase AACT EC 2319 also known as acetoacetylCoA thiolase Thiolase II catalyzes the condensation of two acetylCoA to form 4C compound acetoacetylCoA AcetoacetylCoA is an important starting molecule for biosynthesis of various metabolites Here we report the cDNA cloning and characterization of acetylCoA Cacetyltransferase gene from B monniera The fulllength gene was isolated using a RACE PCR protocol The cDNA encoding AACT was designated as BmAACT FJ947159 revealed an ORF of 1218 bp and 405 amino acids and shares 80  similarity with other plant AACTs Phylogenetic analysis showed that BmAACT is related closely to other dicot plants AACTs The BmAACT gene was overexpressed in Escherichia coli as a 6X Histag fusion protein and purified to homogeneity by NiNTA and gel filtration chromatography Activity of recombinant protein was confirmed by thiolytic cleavage of acetoacetylCoA in the presence of 5 mM Mg2+ showing K m and V max of 2067 μM and 9621 μmol/min respectively with high catalytic efficiency k cat = 230 × 105 min−1 Quantitative realtime PCR analysis showed that the expression of BmAACT is tissuespecific and accumulation of transcripts is greater in roots and petals followed by sepals stem leaf and pedicel

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